Pharmacological and biochemical evidence for the regulation of osteocalcin secretion by potassium channels in human osteoblast-like MG-63 cells

Previous reports have suggested the involvement of voltage-activated calcium (Ca2+) channels in bone metabolism and in particular on the secretion of osteocalcin by osteoblast-like cells,((1)) We now report that potassium (K+) channels can also modulate the secretion of osteocalcin by MG-SS cells, a human osteosarcoma cell line. When 1,25-dihydroxyvitamin D-3(1,25(OH)(2)D-3)-treated MG-63 cells were depolarized by step increases of the extracellular K+ concentration ([K+](out)) from 5-30 mM, osteocalcin (OC) secretion increased from a control value of 218 +/- 13 to 369 +/- 18 ng/mg of protein/48 h (p < 0.005 by analysis of variance), In contrast, in the absence of 1,25(OH)(2)D-3, there is no osteocalcin secretion nor any effect of cell depolarization on this activity, The depolarization-induced increase in 1,25(OH)(2)D-3-dependent osteocalcin secretion was totally inhibited in the presence of 10 mu M Nitrendipine (a Ca2+ channel blocker p < 0.005) without affecting cellular all;aline phosphatase nor cell growth. Charybdotoxin, a selective blocker of Ca2+-dependent K+ channels (maxi-K) present in MG-63 cells,((2)) stimulated 1,25(OH)(2)D-3-induced osteocalcin synthesis about 2-fold (p < 0.005) after either 30, 60, or 120 minute; of treatment, However Charybdotoxin was without effect on basal release of osteocalcin in the absence of 1,25(OH)(2)D-3 pretreatment, Using patch clamp technique, we occasionally observed the presence of a mall conductance K+ channel, compatible with an ATP-dependent KC channel (GK(ATP)) in nonstimulated cells, whereas multiple channel openings were observed when cells were treated with Diazoxide, a sulfonamide derivative which opens GK(ATP). Western blot analysis revealed the presence of the N-terminal peptide of GK(ATP) in MG-63 cells, and its expression was regulated with the proliferation rate of these cells, maximal detection by Western blots being observed during the logarithmic phase of the cycle, Glipizide and Glybenclamide, selective sulfonylureas which can black GK(ATP), dose-dependently enhanced 1,25(OH)(2)D-3-induced OG secretion (p < 0.005), Reducing the extracellular calcium concentration with EGTA (mu M range) totally inhibited the effect of Glipizide and Glybenclamide on osteocalcin secretion (p < 0.005), which remained at the same levels as controls, Diazoxide totally prevented the effect of these sulfonylureas, These results suggest that voltage-activated Ca2+ channels triggered via cell depolarization can enhance 1,25(OH)(2)D-3-induced OC release by MG-63 cells. In addition, OC secretion is increased by blocking two types of K+ channels: maxi-K channels, which normally hyperpolarize cells and close Ca2+ channels, and GK(ATP) channels, The role of these channels is closely linked to the extracellular Ca2+ concentration.